The invention relates to a new polymeric carrier, the method of its preparation and its application in a controlled synthesis of polypeptides.
Biologically active polypeptides may be formed by a stepwise condensation of definite aminoacids in a precisely defined sequence. The classic preparation of such compounds by condensation in solution requires the complex and expensive isolation of reaction products after every reaction step and their characterization. However, this procedure is experimentally unfeasible for peptides with longer chains. Merrifield (P. B. Merrifield; J. Am. Chem. Soc., 85, 2149 (1963)) described a method for overcoming the complications inhering in sequential stepwise condensation of aminoacids. This method consists of three stages -- linking of the first aminoacid to a solid polymer carrier by covalent bond, sequential stepwise reaction of further aminoacids to form the peptide chain, and the eventual cleavage of the formed chain from the carrier and its isolation without damage.
If the growing peptide chain is covalently bound to the solid carrier during the whole sequence of stepwise condensation reactions, separation of products from unreacted agents and products of side reactions is made easy in each reaction step. Complicated isolation operations, which are necessary in the solution process, are reduced to elution or decanting of particles of the solid carrier with anchored growing polypeptide. This procedure simplified the synthesis significantly, considerably shortened the time of synthesis and made its automation possible.
A highly crosslinked copolymer of styrene with divinylbenzene, which is activated for reaction with the first aminoacid, is usually used as the carrier for polypeptide synthesis. This material has two substantial disadvantages limiting its general application. This is, in the first place, the physical structure of the copolymer, which is a xerogel and exhibits therefore a considerable dependence of mechanical properties on the polymer network density. The condensation reactions of aminoacids take place inside the physically homogeneous gel matter and the network density forms a natural limit for molecular weight of synthesized polypeptide. Diffusion coefficients of reaction components, which have to be brought to the reaction site at the growing polypeptide chain, as well as those of reaction products, which have to be washed out after completion of the reaction, are substantially lower in the homogeneous xerogel than in solution. This fact may cause slower establishing of equilibrium in condensation reactions, negatively influence the final degree of conversion in individual reaction steps and, eventually, render more difficult and longer the washing of the material after the elementary condensation reaction. Another disadvantage of the styrene gel follows also from its homogeneously crosslinked structure; namely, it can be used only in solvents swelling the copolymer. Because these are exclusively organic solvents often of low polarity, the choice of reaction possibilities for aminoacid condensation is automatically limited. Application of aqueous reaction systems is impossible. Sorption interacactions of some aminoacids with the nonpolar gel carrier may be expected in the non-aqueous medium, which fact can further render their removal after completion of the condensation step more difficult. All these facts result in non-quantitative course of the individual reaction steps in linking aminoacids and, consequently, some aminoacids are missing in the resulting reaction product after its splitting from the carrier. This can be very unfavorably affect the biological efficiency of the product.
The polymeric carrier according to this invention consists of a hydrophilic gel, which contains besides hydrophilic functional groups also aromatic rings and wherein the primary carrier is further activated by chloromethylation by reaction with formaldehyde and hydrogen chloride. The gel is formed from molecules of hydroxyalkyl acrylates, hydroxyalkyl methacrylates, aminoalkyl acrylates, aminoalkyl methacrylates, acrylamides or methacrylamides linked with a crosslinking monomer of acrylate or methacrylate type and with the third or even further monomer containing aromatic rings in its molecule, which can be activated by chloromethylation. The crosslinking comonomers of acrylate or methacrylate type may be alkylene diacrylates, alkylene dimethacrylates, polyacrylates or polymethacrylates of polyalcohols, dihydroxy- or polyhydroxyethers or alcoholic sugars, alkylene bisacrylamides or divinylbenzene. The object of this invention is further a method for preparation of the gel carrier, which does not possess the negative properties of polystyrene homogeneous gels generally used till now, and application of this gel in controlled synthesis of polypeptides. The carriers are prepared, according to this invention, by a suspension heterogeneous copolymerization of hydrophilic acrylate or methacrylate monomers with the crosslinking comonomer in the presence of a third comonomer containing an aromatic ring in the molecule, as for example, phenyl acrylate, phenyl methacrylate, acrylanilide, methacrylanilide, phenoxyalkyl acrylate or methacrylate, styrene and others. Materials with similar properties may be formed also in a subsequent modification of macroporous suspension copolymers prepared according to Czechoslovak Pat. No. 150819, corresponding to U.S. patent application Ser. No. 281,288, by compounds containing the aromatic ring and forming condensation compounds with the pendent hydroxyl group of the polymer, as for example by aromatic acids, their chlorides or anhydrides, aromatic isocyanates, halogenophenylsilanes, or aromatic epoxy compounds. Finally, the primary carriers can be prepared by polymerization reactions protected by the Czechoslovak Pat. No. 166317 corresponding to British Pat. No. 1,409,967 followed by diazotization of the resulting polymers and coupling with a passive aromatic component. All these copolymers have to be able to undergo chloromethylation to be activated for the reaction with the first aminoacid.
The gels with macroporous structure are formed in the process of heterogeneous suspension copolymerization and are noted for the high mechanical strength and hydrolytic stability. Their specific surface area ranges between 5 and 500 m.sup.2 per gram of the copolymer and they may be prepared in a form of globular particles of uniform size. During the activation, only that portion of the aromatic rings react, which are exposed to the action of formaldehyde and hydrogen chloride on the surface of copolymer. When the material has a large specific surface area, also its capacity is high enough in comparison with homogeneous materials. Then the synthesis of peptide takes place on the solid surface of the copolymer, which does not swell or swells only slightly due to its properties as an aerogel. This permits the use of polar or nonpolar organic solvents as the reaction medium. All complications connected with the slow establishing of reaction equilibria and transport of products from the reaction site, both controlled by diffusion in the gel, are also removed.